Mass spectrometric methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Projects in separation methods and data processing are in progress. In the past year, there has been progress on software development (MassSieve) and isotope labeled fluorescent fusion proteins (published); protein standards and their quantification in collaboration with other laboratories; and ribosomal biochemistry experiments continue.[unreadable] [unreadable] Peptide analyses by LC-MS/MS generate large datasets, imposing labor-intensive efforts to consolidate peptide and/or protein identification information into meaningful knowledge. Software bioinformatics tools can facilitate and accelerate high-throughput peptide data analyses. Toward this goal, we continue refining and testing an integrated workflow designed to maximize the amount of peptide sequence information from LC-MS/MS data. This strategy integrates several independent data search and data mining technologies using a relational database architecture. An iterative search tool acquires results from database search algorithms; spectral filtering and de novo sequencing algorithms are integrated to maximize spectral assignment. The resulting open source software tools (DBParser, MassSieve) provide an automated data processing/analysis environment for researchers. MassSieve is a scalable, multi-platform compatible tool for sorting, comparing and analyzing peptide data with many of the functions of DBParser, processing files from Mascot, Sequest, X!Tandem, and OMSSA. MassSieve uses a faster algorithm to facilitate a more interactive user experience as well as allow the processing of larger datasets. The design of the code is more modular to ease future modification and to allow others to integrate the program into their own laboratory computational environment. In the past year, MassSieve has been distributed as a standalone tool via an open source proteomics website, proteomecommons.org, and fully tested with multiple search engines.[unreadable] [unreadable] After determining the identity of a protein in a mixture by mass spectrometry, quantity is the most important experimental variable required in biological experiments. We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies. Efforts are in progress to create a fusion vector for facile in vitro translation synthesis of GFP linked signature peptides.[unreadable] [unreadable] The ribosome is the universal macromolecular machine that translates the mRNA transcript into polypeptides. Analytical techniques have facilitated the identification of various ribosomal protein isoforms that result from post-translational modifications (PTMs). Beta-methylthioaspartic acid was previously identified as a novel PTM at position 88 in the Escherichia coli ribosomal protein S12. D88 is universal among all S12 bacterial orthologs and mutations at this position are lethal. This unusual PTM has also been identified in the equivalent position of ribosomal protein S12 from phylogenetically distinct bacteria suggesting conservation of the modification. The enzymology of this modification was published recently; our goal is to elucidate the biological function of this novel PTM by identifying specific binding partners. We designed complementary affinity pull-down strategies. The first involves the utilization of a recombinant E. coli S12 protein that has a C-terminal affinity tag. This allows S12 to serve as bait for the identification of proteins that form a stable complex and/or form non-stoichiometric transient interactions. Based on mass spectrometry results we have reproducibly identified and recombinantly tagged 10 candidate S12 binding partners. The second strategy involves creation of synthetic biotinylated peptides that represent the conserved loop region (the putative recognition site) on S12. The peptide pull-down has the advantage of identifying proteins that directly bind or interact with S12. Peptides containing the unmodified and modified Asp 88 serve as baits. After 4 data replicates, we have reproducibly identified the same two translational regulatory proteins that bind to our S12 peptide. These two proteins are among the 10 candidates identified in recombinant S12 pull-downs as potential S12 binders, indicating that the data from the integrated approaches correlate well. We have completed efforts to generate a modified version of the synthetic peptide and are in the process of quantifying binding differences for the modified (containing the PTM) and unmodified peptides. The outcome of these results may indicate the role beta-methylthio-aspartic acid 88 plays for binding specificity of these two proteins.[unreadable] [unreadable] The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. A significant effort of this laboratory is expended in collaboration with the Association of Biomolecular Resource Facilities Proteomics Research Group with regard to qualitative identification and quantification of mixtures. Multi-institutional studies on quantification using stable isotope labeled internal standards are being designed for the coming year.